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Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

Due to the limited options for topical management of skin cancer, this study aimed at developing and evaluating nanoemulsions (NE) for topical delivery of the cytotoxic agent piplartine (piperlongumine). NEs were modified with chitosan or sodium alginate, and the effects on the physicochemical properties, piplartine delivery and formulation efficacy were evaluated. The nanoemulsion droplets displayed similar size (96–112 nm), but opposite charge; the polysaccharides improved piplartine penetration into and across the skin (1.3–1.9-fold) in a similar manner, increasing the ratio “drug in the skin/receptor phase” by 1.4–1.5-fold compared to the plain NE and highlighting their relevance for cutaneous localization. Oleic acid addition to the chitosan-containing NE further increased drug penetration (~1.9–2.0-fold), as did increases in drug content from 0.5 to 1%. The cytotoxicity of piplartine was ~2.8-fold higher when the drug was incorporated in the chitosan-containing NE compared to its solution (IC50 = 14.6 μM) against melanoma cells. The effects of this nanocarrier on 3D melanoma tissues were concentration-related; at 1%, piplartine elicited marked epidermis destruction. These results support the potential applicability of the chitosan-modified nanoemulsion containing piplartine as a new strategy for local management of skin cancer.

Background: Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies. Methods: Evaluated DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation. Results: DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important Metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP-2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2. Conclusion: These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.

Purpose: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance. Experimental Design: Mass spectrometry–based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. Results: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3–mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. Conclusions: Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.

Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability

Ulcerative colitis and Crohn’s disease are chronic inflammatory bowel diseases (IBDs) which burden health systems worldwide; available pharmacological therapies are limited and cost-intensive. Use of peroxisome proliferator activated-receptor γ (PPARγ) ligands for IBD treatment, while promising, lacks solid evidences to ensure its efficacy. Annexin A1 (AnxA1), a glucocorticoid-modulated anti-inflammatory protein, plays a key role on IBD control and is a potential biomarker of IBD progression. We here investigated whether effects of pioglitazone, a PPARγ ligand, rely on AnxA1 actions to modulate IBD inflammation. Experimental colitis was evoked by 2% dextran sodium sulfate (DSS) in AnxA1 knockout (AnxA1−/−) or wild type (WT) C57BL/6 mice. Clinical and histological parameters were more severe for AnxA−/− than WT mice, and 10 mg/kg pioglitazone treatment attenuated disease parameters in WT mice only. AnxA1 expression was increased in tissue sections of diseased WT mice, correlating positively with presence of CD68+ macrophages. Metalloproteinase-9 (MMP-9) and inactive 33 kDa AnxA1 levels were increased in the colon of diseased WT mice, which were reduced by pioglitazone treatment. Cytokine secretion, reactive oxygen species generation and MMP-9 expression caused by lipopolysaccharide (LPS) treatment in AnxA1-expressing RAW 264.7 macrophages were reduced by pioglitazone treatment, effects not detected in AnxA1 knockdown macrophages. LPS-mediated increase of AnxA1 cleaving in RAW 264.7 macrophages was also attenuated by pioglitazone treatment. Finally, pioglitazone treatment increased extracellular signal-regulated kinase (ERK) phosphorylation in AnxA1-expressing RAW 264.7 macrophages, but not in AnxA1-knockdown macrophages. Thus, our data highlight AnxA1 as a crucial factor for the therapeutic actions of pioglitazone on IBDs.

Melanoma is a type of skin cancer with an elevated incidence of metastasis and chemoresistance. Such features hamper treatment success of these neoplasms, demanding the search for new therapeutic options. Using a two-step resin-based approach, we recently demonstrated that cytotoxic prodiginines bind to the inhibitor of apoptosis protein, survivin. Herein, we explore the role of survivin in melanoma and whether its modulation is related to the antimelanoma properties of three cytotoxic prodiginines (prodigiosin, cyclononylprodigiosin, and nonylprodigiosin) isolated from marine bacteria. In melanoma patients and cell lines, survivin is overexpressed, and higher levels negatively impact survival. All three prodiginines caused a decrease in cell growth with reduced cytotoxicity after 24 h compared to 72 h treatment, suggesting that low concentrations promote cytostatic effects in SK-Mel-19 (BRAF mutant) and SK-Mel-28 (BRAF mutant), but not in SK-Mel-147 (NRAS mutant). An increase in G1 population was observed after 24 h treatment with prodigiosin and cyclononylprodigiosin in SK-Mel-19. Further studies indicate that prodigiosin induced apoptosis and DNA damage, as detected by increased caspase-3 cleavage and histone H2AX phosphorylation, further arguing for the downregulation of survivin. Computer simulations suggest that prodigiosin and cyclononylprodigiosin bind to the BIR domain of survivin. Moreover, knockdown of survivin increased long-term toxicity of prodigiosin, as observed by reduced clonogenic capacity, but did not alter short-term cytotoxicity. In summary, prodiginine treatment provoked cytostatic rather than cytotoxic effects, cell cycle arrest at G0/G1 phase, induction of apoptosis and DNA damage, downregulation of survivin, and decreased clonogenic capacity in survivin knockdown cells.

Background Cutaneous melanoma (CM) is the most aggressive subtype of skin cancer, with increasing incidence over the past several decades. DNA methylation is a key element of several biological processes such as genomic imprinting, cell differentiation and senescence, and deregulation of this mechanism has been implicated in several diseases, including cancer. In order to understand the relationship of DNA methylation in CMs, we searched for an epigenetic signature of cutaneous melanomas by comparing the DNA methylation profiles between tumours and benign melanocytes, the precursor cells of CM. Methods We used 20 primary CMs and three primary cell cultures of melanocytes as a discovery cohort. The tumours mutational background was collected as previously reported. Methylomes were obtained using the HM450K DNA methylation assay, and differential methylation analysis was performed. DNA methylation data of CMs from TCGA were recovered to validate our findings. Results A signature of 514 differentially methylated genes (DMGs) was evident in CMs compared to melanocytes, which was independent of the presence of driver mutations. Pathway analysis of this CM signature revealed an enrichment of proteins involved in the binding of DNA regulatory regions (hypermethylated sites), and related to transmembrane signal transducer activities (hypomethylated sites). The methylation signature was validated in an independent dataset of primary CMs, as well as in lymph node and distant metastases (correlation of DNA methylation level: r > 0,95; Pearson’s test: p

Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.

Made available in DSpace on 2020-12-12T01:59:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 A bioassay-guided study aiming at identifying inhibitors of the glycation process on the leaves of Ocotea paranapiacabensis afforded four benzylisoquinoline alkaloids (1-4), with 1 and 2 identified as new naturals products, while 3 and 4 were previously described in the literature, with 3 being identified as magnocurarine. Purification was performed by column chromatography and high-performance liquid chromatography. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, and HRMS. The process of skin aging has been recently associated with advanced glycation end products (AGEs), and strategies inhibiting their formation have been addressed by pharmaceutical companies for the development of novel antiaging compounds. Alkaloids 1-4 were evaluated for their potential to inhibit AGE formation and showed inhibition of 62.9%, 83.3%, 26.1%, and 98.2% (150 μM), respectively. The antiaging potential of compounds 1 and 4 were evaluated with a reconstructed human skin model in vitro, and results showed a decrease in dermis contraction (8.7% and 4.2% respectively for 1 and 4) when compared to the glycated control (57.4%). Additionally, absorption, distribution, metabolism, and excretion (ADME) and toxicity properties were predicted using in silico methods, and the results were considered significantly promising for alkaloids 1 and 4 to continue the development of these alkaloids with skincare properties. Nuclei of Bioassays Biosynthesis and Ecophysiology of Natural Products (NuBBE) Department of Organic Chemistry Institute of Chemistry Saõ Paulo State University (UNESP) Laboratory of Medicinal and Computational Chemistry (LQMC) Centre for Research and Innovation in Biodiversity and Drug Discovery (CIBFar) Institute of Physics of Saõ Carlos University of Saõ Paulo (USP) Federal Institute of Education Science and Technology of Saõ Paulo Rua Stéfano d'Avassi No. 625 Department of Clinical and Toxicological Analysis Faculty of Pharmaceutical Sciences University of Saõ Paulo (USP) Nuclei of Bioassays Biosynthesis and Ecophysiology of Natural Products (NuBBE) Department of Organic Chemistry Institute of Chemistry Saõ Paulo State University (UNESP)

The aim of this study was to further evaluate the antitumoral effect of (PhSe)2-loaded polymeric nanocapsules (NC (PhSe)2) against a resistant melanoma cell line (SK-Mel-103) and develop a xanthan gum-based hydrogel intending the NC (PhSe)2 cutaneous application. For the in vitro evaluation, cells were incubated with free (PhSe)2 or NC (PhSe)2 (0.7–200 μM) and after 48 h the MTT assay, propidium iodide uptake (necrosis marker) and nitrite levels were assessed. The hydrogels were developed by thickening of the NC (PhSe)2 suspension or (PhSe)2 solution with xanthan gum and characterized in terms of average diameter, polydispersity index, pH, drug content, spreadability, rheological profiles and in vitro permeation in human skin. The results showed that NC (PhSe)2 provided a superior antitumoral effect in comparison to free (PhSe)2 (IC50 value of 47.43 μM and 65.05 μM, respectively) and increased the nitrite content. Both compound forms induced propidium iodide uptake, suggesting a necrosis-related pathway could be involved in the cytotoxic action of (PhSe)2. All hydrogels showed pH values around 7, drug content close to the theoretical values (5 mg/g) and mean diameter in the nanometric range. Besides, formulations were classified as non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor. Skin permeation studies revealed that the compound content was higher for the nano-based hydrogel in the dermis layer, demonstrating its superior permeation, achieved by the compound encapsulation. It is the first report on an adequate formulation development for cutaneous application of NC (PhSe)2 that could be used as an adjuvant treatment in melanoma therapy.

Fucoxanthin possesses a well-described antioxidant activity that might be useful for human skin photoprotection. However, there is a lack of scientific information regarding its properties when applied onto human skin. Thus, the objective of the present study was to assess the photoprotective and phototoxicity potential of fucoxanthin based on its ultraviolet (UVB 280&ndash, 320 nm, UVA 320&ndash, 400 nm) and visible (VIS 400&ndash, 700 nm) absorption, photostability, phototoxicity in 3T3 mouse fibroblast culture vs. full-thickness reconstructed human skin (RHS), and its ability to inhibit reactive oxygen species formation that is induced by UVA on HaCaT keratinocytes. Later, we evaluated the antioxidant properties of the sunscreen formulation plus 0.5% fucoxanthin onto RHS to confirm its bioavailability and antioxidant potential through the skin layers. The compound was isolated from the alga Desmarestia anceps. Fucoxanthin, despite presenting chemical photo-instability (dose 6 J/cm2: 35% UVA and 21% VIS absorbance reduction), showed acceptable photodegradation (dose 27.5 J/cm2: 5.8% UVB and 12.5% UVA absorbance reduction) when it was added to a sunscreen at 0.5% (w/v). In addition, it increased by 72% of the total sunscreen UV absorption spectra, presenting UV-booster properties. Fucoxanthin presented phototoxic potential in 3T3 fibroblasts (mean photo effect 0.917), but it was non-phototoxic in the RHS model due to barrier function that was provided by the stratum corneum. In addition, it showed a significant inhibition of ROS formation at 0.01% (p &lt, 0.001), in HaCat, and in a sunscreen at 0.5% (w/v) (p &lt, 0.001), in RHS. In conclusion, in vitro results showed fucoxanthin protective potential to the skin that might contribute to improving the photoprotective potential of sunscreens in vivo.

The development of products for topical applications requires analyses of their skin effects before they are destined for the market. At present, the ban on animal use in several tests makes the search for in vitro models (such as artificial skin) necessary to characterize the risks involved. In this work, tissue engineering concepts were used to manufacture collagen-free three-dimensional scaffolds for cell growth and proliferation. Two different human skin models&mdash, reconstructed human epidermis and full-thickness skin&mdash, were developed from electrospun scaffolds using synthetic polymers such as polyethylene terephthalate, polybutylene terephthalate, and nylon 6/6. After the construction of these models, their histology was analyzed by H&amp, E staining and immunohistochemistry. The results revealed a reconstructed epidermal tissue, duly stratified, obtained from the nylon scaffold. In this model, the presence of proteins involved in the epidermis stratification process (cytokeratin 14, cytokeratin 10, involucrin, and loricrin) was confirmed by immunohistochemistry and Western blot analysis. The nylon reconstructed human epidermis model&rsquo, s applicability was evaluated as a platform to perform irritation and corrosion tests. Our results demonstrated that this model is a promising platform to assess the potential of dermal irritation/corrosion of chemical products.

Background: Osteoarthritis (OA) is a progressive and multifactorial disease that is associated with aging. A number of changes occur in aged cartilage, such as increased oxidative stress, decreased markers of healthy cartilage, and alterations in the autophagy pathway. Propolis extracts contain a mixture of polyphenols and it has been proved that they have high antioxidant capacity and could regulate the autophagic pathway. Our objective was to evaluate the effect of ethanolic extract of propolis (EEP) on chondrocytes that were stimulated with IL-1&beta, Methods: Rabbit chondrocytes were isolated and stimulated with IL-1&beta, and treated with EEP. We evaluated cell viability, nitric oxide production, healthy cartilage, and OA markers, and the expression of three proteins associated with the autophagy pathway LC3, ATG5, and AKT1. Results: The EEP treatment reduces the expression of LC3, ATG5, and AKT1, reduces the production of nitric oxide, increases the expression of healthy markers, and reduces OA markers. Conclusions: These results suggest that treatment with EEP in chondrocytes that were stimulated with IL-1&beta, has beneficial effects, such as a decrease in the expression of proteins associated with autophagy, MMP13, and production of nitric oxide, and also increased collagen II.

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased theΔψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

Three-dimensional skin models, also named 3D skin models, human skin equivalents (HSEs), or Human Epidermal Equivalents (HEEs), have been increasingly used for chemical assessments in terms of efficacy and safety. Considering this, we developed an HEE model using immortalized HaCaT cells, aiming to overcome the limitation of primary tissue source. Our 3D model (HaCaT-HEE) exhibited important markers of cell differentiation (CK10, CK14, involucrin, and filaggrin), although the stratum corneum was shown to be modest. Besides, the model showed a good prediction potential considering membrane permeability, sensitivity, specificity, and accuracy in distinguishing irritant and corrosive effects after exposure to selected chemicals recommended by the OECD protocols. We also validated the formazan determination for the MTT method using High-Performance Liquid Chromatography (HPLC). For that, we considered carry over, linearity, reproducibility/robustness, accuracy, precision, selectivity, and matrix effect, according to the Food and Drug Administration (FDA) guideline. Based on our results, we can conclude that our model has an acceptable predictive value for the safety evaluation of compounds after skin exposure, with the great advantage of being constructed using immortalized cells.

Skin model cultivation under static conditions limits the observation of the toxicity to this single organ. Biology-inspired microphysiological systems associating skin with a liver in the same circulating medium provide a more comprehensive insight into systemic substance toxicity; however, its advantages or limitations for topical substance toxicity remain unknown. Herein, we performed topical (OECD test guideline no. 439) and systemic administration of terbinafine in reconstructed human skin (RHS) vs. a RHS plus liver model cultured in TissUse' HUMIMIC Chip2 (Chip2). Aiming for a more detailed insight into the cutaneous substance irritancy/toxicity, we assessed more than the MTT cell viability: lactate dehydrogenase (LDH), lactate and glucose levels, as well as inherent gene expressions. Sodium dodecyl sulfate (SDS) was the topical irritant positive control. We confirmed SDS irritancy in both static RHS and Chip2 culture by the damage in the morphology, reduction in the lactate production and lower glucose consumption. In the static RHS, the SDS-treated tissues also released significantly high LDH (82%; p 0.05) and significantly lower IL-6 release (p 0.05), corroborating with the other metabolic levels. In both static RHS and Chip2 conditions, we confirmed absence of irritancy or systemic toxicity by LDH, glucose or lactate levels for topical 1% and 5% terbinafine and systemic 0.1% terbinafine treatment. However, topical 5% terbinafine treatment in the Chip2 upregulated IL-1α in the RHS, unbalanced apoptotic and proliferative cell ratios in the liver and significantly increased its expression of CYP1A2 and 3A4 enzymes (p 0.05), proving that it has passed the RHS barrier promoting a liver impact. Systemic 0.1% terbinafine treatment in the Chip2 increased RHS expression of EGFR, increased apoptotic cells in the liver, downregulated liver albumin expression and upregulated CYP2C9 significantly (p 0.05), acting as an effective hepatotoxic terbinafine control. The combination of the RHS and liver model in the Chip2 allowed a more sensitive assessment of skin and hepatic effects caused by chemicals able to pass the skin (5% terbinafine and SDS) and after systemic 0.1% terbinafine application. The present study opens up a more complex approach based on the microphysiological system to assess more than a skin irritation process.

Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients' biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.

The hair-dyeing ingredient, p-phenylenediamine (PPD), was previously reported to be mutagenic, possibly by inducing oxidative stress. However, the exact mechanism of PPD in inducing oxidative stress upon skin exposure during hair-dyeing in human keratinocytes remains unknown. The aim of our studies was therefore to investigate the toxicity of PPD and its by-products in human immortalized keratinocytes (HaCaT) after auto-oxidation and after reaction with hydrogen peroxide (H2O2). We found that the PPD half maximal effective cytotoxic concentration (EC50) to HaCaT is 39.37 and 35.63 μg/mL after 24 and 48 h, respectively, without addition of H2O2 to induce oxidation. When PPD (10 or 100 μg/mL) is combined with 10.5 μg/mL of H2O2, intracellular ROS production by HaCaT after 1 h was significantly increased and enhanced levels of DNA damage were observed after 4 h of exposure. After 24 h incubations, 20 μg/mL of PPD increased the level of DNA oxidation in HaCaT. Also, we found that the in vitro reaction between PPD and H2O2, even below the maximum allowance by cosmetic industries, released hydroxyl radicals which can damage DNA. Taken together, we conclude that PPD alone and when combined with H2O2 increases the formation of reactive oxygen species in human keratinocytes, leading to oxidative stress and subsequent DNA damage. These alterations suggest that the mechanism by which PPD exposure, alone or combined with H2O2, damages keratinocytes by the formation of the high reactive HO∙ radicals.

It is generally accepted that three-dimensional (3D) cell culture systems better represent cell physiology and morphology than two-dimensional (monolayer) cell cultures. The aim of this work was to develop a 3D model that could efficiently evaluate polymeric materials biocompatibility using polybutylcyanoacrylate nanoparticles (PBCA-NP). Normal human fibroblasts and melanoma cells were cultured both in monolayer and on type I collagen matrix. Trypan blue and MTT assays were used to evaluate PBCA-NP cytotoxicity, which was shown to be dose- and time-dependent to all cell types. These results were confirmed by cell death analysis with flow cytometry. There is evidence that treatment with PBCA-NP in concentrations close to the IC50 inhibit autophagy in fibroblasts and melanoma cells when cultured in monolayer, but this response could not be observed in the 3D system. These findings strongly suggest that PBCA-NP can modify cell response depending on the concentration used and the conditions of cell culture.

Gliomas account for the majority of primary malignant brain tumors and present invasive behavior into adjacent healthy tissue. While 4-NC had previously shown to induce apoptotic cell death in a melanoma model, for the glioma model described in this paper 4-NC is cytotoxic for the cells with the induction of the autophagic pathway. Trypan blue exclusion assay showed that 4-NC was cytotoxic in a dose-dependent manner for A172 and T98G cell lines. IC10 and IC50 values were at 32 µM and 41 µM for A172 and T98G respectively. Inhibition of cell proliferation was observed by total cell counts and by cell cycle analysis by flow cytometry, with cell cycle arrest of A172 and T98G cell lines respectively in the G1/G0 and S phases of the cell cycle. 4-NC induced up-regulation of autophagic pathways, as shown by immunoblotting for LC3-I/II, Real-Time PCR for ATG-7 and Beclin-1 genes, and by fluorescence microscopy observation of autophagic vacuoles in cells transfected with GFP-LC3 and electron microscopy. Glioma cells concomitantly treated with 4-NC and 3-MA, an inhibitor of the autophagic process, are more sensible to cell death, suggesting that autophagy protects the cells from the action of 4-NC.

Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (312s). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products. Because of the complex reaction, this cytotoxic mechanism has not yet been elucidated and is the subject of this study. Here, we topically exposed p-phenylenediamine (PPD), Resorcinol (RES), Hydrogen Peroxide (H2O2) alone or as a mixture to RhE and evaluated parameters related to skin irritation such as epidermal' viability, keratinocytes damage, barrier loss and IL-1 alpha. Our data indicates that ingredients tested alone did not lead to an increase of cytotoxic parameters related to skin irritation. However, when the mixture of PPD/H2O2/RES and PPD/H2O2 was applied to the RhE, some of the parameters such as morphological changes including the presence of apoptotic cells, barrier loss and increased IL-1 alpha release were observed. The results indicate that the mixture of ingredients used in permanent hair dyes have an irritant effect in RhE while the ingredients alone not.

Currently, there is a strong global trend towards the development of in vitro models to replace the use of animals in safety evaluation tests. Reconstructed Human Epidermis (RHE) models have been employed as an alternative method to animal testing of skin corrosion and irritation potential of chemical compounds. However, the consequences of an absence of the dermal compartment in these models should be considered since the cross-talk between fibroblasts and keratinocytes is fundamental for promoting proper epidermal stratification, homeostasis, inflammatory response and wound healing. In this study, we compare in-house developed models of Reconstructed Human Epidermis (i.e. USP-RHE) and full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested (2-phenylethyl bromide, benzylacetone, lactic acid, octanoic acid) as corrosive or non-corrosive. Furthermore, we have demonstrated higher cell viability of the USP-FTS model compared to the USP-RHE model, a sign of its improved barrier function, following the exposure to the substances test on the corrosion assay. This emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances.

Melanoma is a highly invasive and metastatic cancer with high mortality rates and chemoresistance. Around 50% of melanomas are driven by activating mutations in BRAF that has led to the development of potent anti-BRAF inhibitors. However resistance to anti-BRAF therapy usually develops within a few months and consequently there is a need to identify alternative therapies that will bypass BRAF inhibitor resistance. The curcumin analogue DM-1 (sodium 4-[5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate) has substantial anti-tumor activity in melanoma, but its mechanism of action remains unclear. Here we use a synthetic lethal genetic screen in Saccharomyces cerevisiae to identify 211 genes implicated in sensitivity to DM-1 toxicity. From these 211 genes, 74 had close human orthologues implicated in oxidative phosphorylation, insulin signaling and iron and RNA metabolism. Further analysis identified 7 target genes (ADK, ATP6V0B, PEMT, TOP1, ZFP36, ZFP36L1, ZFP36L2) with differential expression during melanoma progression implicated in regulation of tumor progression, cell differentiation, and epithelial-mesenchymal transition. Of these TOP1 and ADK were regulated by DM-1 in treatment-naive and vemurafenib-resistant melanoma cells respectively. These data reveal that the anticancer effect of curcumin analogues is likely to be mediated via multiple targets and identify several genes that represent candidates for combinatorial targeting in melanoma.

Considering that tumor development is generally multifactorial, therapy with a combination of agents capable of potentiating cytotoxic effects is promising. In this study, we co-encapsulated C6 ceramide (0.35%) and paclitaxel (0.50%) in micro and nanoemulsions containing tributyrin (a butyric acid pro-drug included for potentiation of cytotoxicity), and compared their ability to co-localize the drugs in viable skin layers. The nanoemulsion delivered 2- and 2.4-fold more paclitaxel into viable skin layers of porcine skin in vitro at 4 and 8h post-application than the microemulsion, and 1.9-fold more C6 ceramide at 8h. The drugs were co-localized mainly in the epidermis, suggesting the nanoemulsion ability for a targeted delivery. Based on this result, the nanoemulsion was selected for evaluation of the nanocarrier-mediated cytotoxicity against cells in culture (2D model) and histological changes in a 3D melanoma model. Encapsulation of the drugs individually decreased the concentration necessary to reduce melanoma cells viability to 50% (EC50) by approximately 4- (paclitaxel) and 13-fold (ceramide), demonstrating an improved nanoemulsion-mediated drug delivery. Co-encapsulation of paclitaxel and ceramide further decreased EC50 by 2.5-4.5-fold, and calculation of the combination index indicated a synergistic effect. Nanoemulsion topical administration on 3D bioengineered melanoma models for 48h promoted marked epidermis destruction, with only few cells remaining in this layer. This result demonstrates the efficacy of the nanoemulsion, but also suggests non-selective cytotoxic effects, which highlights the importance of localizing the drugs within cutaneous layers where the lesions develop to avoid adverse effects.

Made available in DSpace on 2018-11-26T17:16:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Tympanic membrane perforations are due to common otologic problems. The current treatments to heal tympanic membrane perforation, such as myringoplasty, have some disadvantages, including the need for autologous grafting, which is rapidly absorbed by the organism before perforation recovery is complete. To improve the structural and functional tympanic membrane healing after surgery, we propose a new branch of artificial grafts. In this study, we report the development of artificial grafts using electrospun bioabsorbable polymers. Polymers such as poly (L-lactic acid) and poly (lactic-co-glycolic acid) acted as the scaffold for cell growth in a co-culture of fibroblasts and keratinocytes. This co-culture promoted the growth of an epithelial-equivalent tissue over the electrospun scaffold, which was used as an alternative graft in myringoplasty. The in vivo study was performed in Sprague Dawley rats. Ear endoscopy was performed 30 days after surgery and showed that tympanic membrane perforations treated with artificial grafts healed naturally, completely and with the possibility of maintaining their actual functionality. In conclusion, our study described a new artificial graft created specifically to fulfill the requirements of perforated tympanic membrane healing processes, which are compatibility, proper durability and less intense side effects following myringoplasty. (C) 2016 Elsevier B.V. All rights reserved. Univ Sao Paulo, Inst Quim, Dept Quim Fundamental, Sao Paulo, Brazil Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias Botucatu, Dept Morfol, Botucatu, SP, Brazil Complex Hosp Edmundo Vasconcelos, Ctr Otorrinolaringol & Fonoaudiol Sao Paulo, Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias Botucatu, Dept Morfol, Botucatu, SP, Brazil FAPESP: 2011/21442-6 FAPESP: 2011/18017-1 FAPESP: 2011/22812-1 CNPq: 303030/2013-5 CNPq: 400145/2014-6

This study investigates the mechanism of action behind the long-term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, the main etiologic factor for skin carcinogenesis. We hypothesized that carbaryl exposure may increase deleterious effects of UV solar radiation on skin melanocytes. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100 μM) and solar radiation (375 mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of CDKN1A, BRCA1/2 and MDM2 genes was notably more intense in the combined treatment group, in a synergistic manner. Flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation.

Skin aging is a natural process of the human body that may be accelerated due to extrinsic causes. Libidibia ferrea, popularly known as juca, is a small tree, which possesses an abundant phenolic composition with potential antioxidant and enzymatic inhibition activities. Thus, this work aimed to investigate the anti-wrinkle and anti-whitening potentials of juca trunk bark (LFB) and pod (LFP) extracts. A comprehensive analysis of LFB and LFP phenolic composition was accomplished by means of liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Effects on skin degradation were assessed by inhibitory enzymatic activity against elastase, hyaluronidase and collagenase through colorimetric assays. Cellular viability in B16F10 and primary fibroblasts were determined by Trypan Blue exclusion assay. Anti-melanogenic effects on B16F10 cells were evaluated using cellular tyrosinase, melanin content, western blot, and RT-qPCR analyses. Inhibition of matrix metalloproteinase-2 and metalloproteinase-9 (MMP-2 and MMP-9) was determined by gelatin zymography and western blot methodologies. LC–MS/MS analyses of LFB and LFP extracts allowed the characterization of 18 compounds, among them, flavonoids, phenolic acids, and secoridoids. Additionally the pod and trunk bark compositions were compared. Hyaluronidase inhibitory activity for both extracts, LFB (IC50 = 8.5 ± 0.8 µg/mL) and LFP (IC50 = 16 ± 0.5 µg/mL), was stronger than standard rutin (IC50 = 27.6 ± 0.06). Pro-MMP-2 was significantly inhibited by both extracts. LFB and LFP decreased the melanin content in B16F10 due to tyrosinase inhibitory activity. L. ferrea extracts has high potential as a cosmetic ingredient due to its anti-wrinkle and depigmentant effects.

Indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO1 is a leading target for interventions aimed at restoring melanoma immune surveillance. Here, in a scenario involving the tryptophan catabolism, we report that melatonin biosynthesis is driven by 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO1, in human fibroblasts, melanocytes and melanoma cells. In addition to melatonin biosynthesis, 1-MT induced the expression of tryptophan hydroxylase, arylalkylamine-N-acetyltransferase and hydroxyindole O-methyltransferase mRNA in fibroblasts and melanocytes. We observed a great variability in the levels of IDO1 mRNA expression and kynurenine release between skin cells and melanoma cell lines in response to interferon-γ, a classical IDO1 inducer. In this setting, melatonin was shown to downregulate kynurenine production. Furthermore, in a condition of low basal activity of IDO1, it was observed that 1-MT, as well melatonin, inhibited the proliferation of human melanoma cells. Taken together, our results suggest that 1-MT may serve as more than just a tool to disrupt tumor immune escape (via the inhibition of IDO1) because it was shown to act directly on the proliferation of human melanoma cells and induce melatonin biosynthesis in the tumor milieu. Moreover, 1-MT-mediated inhibition of IDO occurs in normal skin and melanoma cells, which addresses the possibility that all cells in the skin microenvironment can be targeted by 1-MT. Our findings provide innovative approaches into understanding tumor therapy related to the control of tryptophan metabolism by 1-MT.

Malignant melanoma is a highly aggressive form of skin cancer with a high mortality rate if not discovered in early stages. Although a limited number of treatment options for melanoma currently exist, patients with a more aggressive form of this cancer frequently decline treatment. DM-1 is a sodium phenolate and curcumin analog with proven anticancer, anti-proliferative and anti-metastatic properties. In this paper, the DM-1 compound showed in vivo antitumor activity alone or in combination with chemotherapeutic DTIC in B16F10 melanoma-bearing mice. Beneficial effects such as melanoma tumor burden reduction with pyknotic nuclei, decreased nuclei/cytoplasmic ratio and nuclear degradation occurred after DM-1 treatment. No toxicological changes were observed in the liver, kidneys, spleen and lungs after DM-1 monotherapy or DTIC combined therapy. DTIC+DM-1 treatment induced the recovery of anemia arising from melanoma and immunomodulation. Both DM-1 treatment alone and in combination with DTIC induced apoptosis with the cleavage of caspase-3, -8 and -9. Furthermore, melanoma tumors treated with DM-1 showed a preferential apoptotic intrinsic pathway by decreasing Bcl-2/Bax ratio. Considering the chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, DM-1 compound in monotherapy or in combination therapy provides a promising improvement in melanoma treatment with a reduction of side effects.

Strategies for patients whose melanomas are BRAF wild-type are completely lacking. We here report on the mechanisms underlying the long-term responses (>12 months) of 2 BRAF wild-type (WT) melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. A panel of 7 BRAF/NRAS-WT cell lines was leveraged to investigate strategies to overcome chemoresistance. Electron microscopy, western blotting and flow cytometry assays were integrated to simultaneously follow fluctuations in autophagic flux, reactive oxygen species levels and cytotoxicity caused from chronic treatment of cells with chemotherapy, MK-2206, lysomotropic agents and/or anti-oxidants. MK-2206 potently inhibited phospho-AKT signaling, but did not impact melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin induced little apoptosis following 3-day treatment, but led to cytotoxicity in long-term colony formation and 3D spheroid assays. Mechanistically the combination was noted to induce autophagy and disrupt reactive oxygen species homeostasis, as shown by increased LC3-II expression, the formation of LC3 punctae, acridine orange protonation, the accumulation of autophagosomes and increased ROS detection using DHR-123. At earlier time points, autophagy was protective with cytotoxicity enhanced following chloroquine and bafilomycin treatment as well as the increased cytotoxic effects seen in ATG5-/- MEFs. Although prolonged MK-2206/chemotherapy treatment (6-9 days) led to high levels of autophagy induction and a switch to caspase-dependent apoptosis, drug resistant clones still eventually emerged. The importance of autophagy for the escape process was suggested by the lack of ATG5-/- MEF colonies seen following combination therapy treatment. Autophagy inhibition enhanced the cell death response through increased generation of ROS and could be reversed by anti-oxidants. In summary, we demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma. We further show that the administration of an autophagy inhibitor may prevent resistance from emerging to this drug combination. Citation Format: Vito W. Rebecca, Renato Massaro, Inna Fedorenko, Geoffrey T. Gibney, Vernon K. Sondak, Ravi K. Amaravadi, Shengkan Jin, Silvya S. Maria-Engler, Ragini Kudchadkar, Keiran S. M. Smalley. BRAF wild-type melanoma survival hinges upon AKT activity and adaptive autophagic reprogramming in response to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2014-809

Although targeted therapy strategies are currently lacking for patients whose melanomas are BRAF wild-type, some therapeutic efficacy was noted in a phase I clinical trial of the AKT inhibitor MK-2206 in combination with Carboplatin (C) and Paclitaxel (P). In the present study we addressed the mechanism underlying the responses to MK-2206 + CP in BRAF wild-type melanoma. As single agents, MK-2206, carboplatin and paclitaxel had only limited cytotoxic activity in both 2D and 3D cell culture and induced very little apoptosis. Prolonged treatment (>9 days) with the AKT inhibitor in combination with C+P led to significant levels of cell death in 4 3D spheroid culture models of BRAF-wild type melanoma. In contrast, treatment with single agent MK-2206 or C+P alone induced little cell death. To investigate the mechanism of interaction between these three compounds we focused upon autophagy induction. It was noted that significant levels of autophagy, as demonstrated by increased LC3-II staining, p62 expression and increased acridine orange staining, were induced following up to 6 days of treatment with the combination. In this context autophagy was protective, with levels of cell death induced by the combination significantly enhanced following treatment with the autophagy inhibitor chloroquine. The protective autophagy induced by MK-2206 + CP was only transient, with diminished levels of autophagy being observed by 9 days. The observed reduction in autophagic flux was associated with marked increases in cell death. In summary it appears that chronic insult with MK-2206 and CP pushes autophagy to its limit, relinquishing its protective role and enhancing sensitivity of the cells to therapy-induced apoptosis. These studies form the basis for a phase II trial evaluating MK-2206 in combination with chemotherapy, as well as autophagy inhibitors, in patients whose melanomas are BRAF-wild type. Citation Format: Vito W. Rebecca, Renato R. Massaro, Silvya S. Maria-Engler, Jane L. Messina, Geoffrey T. Gibney, Vernon K. Sondak, Ragini Kudchadkar, Keiran S. Smalley. Exhaustion of autophagy-mediated cell survival underlies the pre-clinical and clinical activity of chemotherapy combined with the AKT inhibitor MK-2206 in BRAF wild-type melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2013-1659